The present invention relates to prognostic methods which are useful in medicine, particularly cancer chemotherapy. More particularly, the invention relates to assessment of tumor cell gene expression in a patient. The resistance of tumor cells to chemotherapeutic agents that target DNA, especially agents that damage DNA in the manner of platinating agents is assayed by examining the mRNA expressed from genes involved in DNA repair in humans.
Cancer arises when a normal cell undergoes neoplastic transformation and becomes a malignant cell. Transformed (malignant) cells escape normal physiologic controls specifying cell phenotype and restraining cell proliferation. Transformed cells in an individual""s body thus proliferate, forming a tumor. When a tumor is found, the clinical objective is to destroy malignant cells selectively while mitigating any harm caused to normal cells in the individual undergoing treatment.
Chemotherapy is based on the use of drugs that are selectively toxic (cytotoxic) to cancer cells. Several general classes of chemotherapeutic drugs have been developed, including drugs that interfere with nucleic acid synthesis, protein synthesis, and other vital metabolic processes. These generally are referred to as antimetabolite drugs. Other classes of chemotherapeutic drugs inflict damage on cellular DNA. Drugs of these classes generally are referred to as genotoxic. Susceptibility of an individual neoplasm to a desired chemotherapeutic drug or combination of drugs often, however, can be accurately assessed only after a trial period of treatment. The time invested in an unsuccessful trial period poses a significant risk in the clinical management of aggressive malignancies.
The repair of damage to cellular DNA is an important biological process carried out by a cell""s enzymatic DNA repair machinery. Unrepaired lesions in a cell""s genome can impede DNA replication, impair the replication fidelity of newly synthesized DNA and/or hinder the expression of genes needed for cell survival. Thus, genotoxic drugs generally are considered more toxic to actively dividing cells that engage in DNA synthesis than to quiescent, nondividing cells. Normal cells of many body tissues are quiescent and commit infrequently to re-enter the cell cycle and divide. Greater time between rounds of cell division generally is afforded for the repair of DNA damage in normal cells inflicted by chemotherapeutic genotoxins. As a result, some selectivity is achieved for the killing of cancer cells. Many treatment regimens reflect attempts to improve selectivity for cancer cells by coadministering chemotherapeutic drugs belonging to two or more of these general classes.
Because effective chemotherapy in solid tumors usually requires a combination of agents, the identification and quantification of determinants of resistance or sensitivity to each single drug has become an important tool to design individual combination chemotherapy.
Two widely used genotoxic anticancer drugs that have been shown to damage cellular DNA are cisplatin (DDP) and carboplatin. Cisplatin and/or carboplatin currently are used in the treatment of selected, diverse neoplasms of epithelial and mesenchymal origin, including carcinomas and sarcomas of the respiratory, gastrointestinal and reproductive tracts, of the central nervous system, and of squamous origin in the head and neck. Cisplatin in combination with other agents is currently preferred for the management of testicular carcinoma, and in many instances produces a lasting remission. (Loehrer et al., 1984, 100 Ann.Int. Med. 704). Cisplatin (DDP) disrupts DNA structure through formation of intrastrand adducts. Resistance to platinum agents such as DDP has been attributed to enhanced tolerance to platinum adducts, decreased drug accumulation, or enhanced DNA repair. Although resistance to DDP is multifactoral, alterations in DNA repair mechanisms probably play a significant role.
The glutathione-S-transferase (GST) family of proteins is involved in detoxification of cytotoxic drugs. By catalyzing the conjugation of toxic and carcinogenic electrophilic molecules with glutathione the GST enzymes protect cellular macromolecules from damage (Boyer et al., Preparation, characterization and properties of glutathione S-transferases. In: Zakim D, Vessey D (eds.) Biochemical Pharmacology and Toxicology. New York, N.Y.: John Wiley and Sons, 1985.). A certain isomeric type of these proteins, the glutathione S-transferase Pi (GST-pi, also to be interchangeably refered to as GSTP1 or GST-xcfx80 herein) is widely expressed in human epithelial tissues and has been demonstrated to be over-expressed in several tumors (Terrier et al., Am J Pathol 1990; 137: 845-853; Moscow et al., Cancer Res 1989; 49: 1422-1428). Increased GST-pi levels have been found in drug resistant tumors, although the exact mechanism remains unclear (Tsuchida et al., Crit Rev Biochem Mol Biol 1992; 27: 337-384). Previous studies have suggested that low expression of GST protein (not mRNA) is associated with response to platinum-based chemotherapy (Nishimura et al., Cancer. Clin Cancer Res 1996; 2:1859-1865; Tominaga, et al., Am. J. Gastro. 94:1664-1668, 1999; Kase, et al., Acta Cytologia. 42: 1397-1402, 1998). However, these studies did not measure quantitative gene expression, but used a semi-quantitative immunohistochemical staining method to measure protein levels. However, quantitative GST-pi gene expression measurements are needed to achieve a very effective prognostication.
Most pathological samples are routinely fixed and paraffin-embedded (FPE) to allow for histological analysis and subsequent archival storage. Thus, most biopsy tissue samples are not useful for analysis of gene expression because such studies require a high integrity of RNA so that an accurate measure of gene expression can be made. Currently, gene expression levels can be only qualitatively monitored in such fixed and embedded samples by using immunohistochemical staining to monitor protein expression levels.
Until now, quantitative gene expression studies including those of GST-pi expression have been limited to reverse transcriptase polymerase chain reaction (RT-PCR) amplification of RNA from fresh or frozen tissue.
The use of frozen tissue by health care professionals poses substantial inconveniences. Rapid biopsy delivery to avoid tissue and subsequent mRNA degradation is the primary concern when planning any RNA-based quantitative genetic marker assay. The health care professional performing the biopsy, must hastily deliver the tissue sample to a facility equipped to perform an RNA extraction protocol immediately upon tissue sample receipt. If no such facility is available, the clinician must promptly freeze the sample in order to prevent mRNA degradation. In order for the diagnostic facility to perform a useful RNA extraction protocol prior to tissue and RNA degradation, the tissue sample must remain frozen until it reaches the diagnostic facility, however far away that may be. Maintenance of frozen tissue integrity during transport using specialized couriers equipped with liquid nitrogen and dry ice, comes only at a great expense.
Routine biopsies generally comprise a heterogenous mix of stromal and tumorous tissue. Unlike with fresh or frozen tissue, FPE biopsy tissue samples are readily microdissected and separated into stromal and tumor tissue and therefore, offer andvantage over the use of fresh or frozen tissue. However, isolation of RNA from fixed tissue, and especially fixed and paraffin embedded tissue, results in highly degraded RNA, which is generally not applicable to gene expression studies.
A number of techniques exist for the purification of RNA from biological samples, but none is reliable for isolation of RNA from FPE samples. For example, Chomczynski (U.S. Pat. No. 5,346,994) describes a method for purifying RNA from tissues based on a liquid phase separation using phenol and guanidine isothiocyanate. A biological sample is homogenized in an aqueous solution of phenol and guanidine isothiocyanate and the homogenate thereafter mixed with chloroform. Following centrifugation, the homogenate separates into an organic phase, an interphase and an aqueous phase. Proteins are sequestered in the organic phase, DNA in the interphase, and RNA in the aqueous phase. RNA can be precipitated from the aqueous phase. Unfortunately, this method is not applicable to fixed and paraffin-embedded (FPE) tissue samples.
Other known techniques for isolating RNA typically utilize either guanidine salts or phenol extraction, as described for example in Sambrook, J. et al., (1989) at pp. 7.3-7.24, and in Ausubel, F. M. et al., (1994) at pp. 4.0.3-4.4.7. Again, none of the known methods provides reproducible quantitative results in the isolation of RNA from paraffin-embedded tissue samples.
Techniques for the isolation of RNA from paraffin-embedded tissues are thus particularly needed for the study of gene expression in tumor tissues, since expression levels of certain receptors or enzymes can be used to determine the likelihood of success of a particular treatment.
There is a need for a method of quantifying GST-pi mRNA from paraffinized tissue in order to provide an early prognosis for proposed genotoxic cancer therapies. As a result, there has been a concerted yet unsuccessful effort in the art to obtain a quantification of GST-pi expression in fixed and paraffinized (FPE) tissue. Accordingly, it is the object of the invention to provide a method for assessing GST-pi levels in tissues fixed and paraffin-embedded (FPE) and prognosticate the probable resistance of a patient""s tumor to treatment with DNA damaging agents, creating the type of lesions in DNA that are created by DNA platinating agents, by examination of the amount of GST-pi mRNA in a patient""s tumor cells and comparing it to a predetermined threshold expression level.
In one aspect of the invention there is provided a method for assessing levels of expression of GST-pi mRNA obtained from fixed and paraffin-embedded (FPE) fixed and paraffin-embedded (FPE) tumor cells.
In another aspect of the invention there is provided a method of quantifying the amount of GST-pi mRNA expression relative to an internal control from a fixed and paraffin-embedded (FPE) tissue sample. This method includes isolation of total mRNA from said sample and determining the quantity of GST-pi mRNA relative to the quantity of an internal control gene""s mRNA.
In an embodiment of this aspect of the invention, there are provided oligonucleotide primers having the sequence of GST-F (SEQ ID NO: 1) or GST-R (SEQ ID NO:2) and sequences substantially identical thereto. The invention also provides for oligonucleotide primers having a sequence that hybridizes to SEQ ID NO: 1 or SEQ ID NO:2 or their complements under stringent conditions.
In yet another aspect of the invention there is provided a method for determining a chemotherapeutic regimen for a patient, comprising isolating RNA from a fixed and paraffin-embedded (FPE) tumor sample; determining a gene expression level of GST-pi in the sample; comparing the GST-pi gene expression levels in the sample with a predetermined threshold level for the GST-pi gene; and determining a chemotherapeutic regimen based on results of the comparison of the GST-pi gene expression level with the predetermined threshold level.
The invention further relates to a method of normalizing the uncorrected gene expression (UGE) of GST-pi relative to an internal control gene in a tissue sample analyzed using TaqMan(copyright) technology to known GST-pi expression levels relative to an internal control from samples analyzed by pre-TaqMan(copyright) technology.